HuMSC-EV induce monocyte/macrophage mobilization to orchestrate neovascularization in wound healing process following radiation injury

This study aims to investigate the mechanisms of human mesenchymal stem cell-derived extracellular vesicles (HuMSC-EV)-induced proangiogenic paracrine effects after radiation injury. HuMSC-EV were locally administered in mice hindlimb following 80-Gy X-ray irradiation and animals were monitored at different time points. HuMSC-EV improved neovascularization of the irradiated tissue, by stimulating angiogenesis, normalizing cutaneous blood perfusion, and increasing capillary density and production of proangiogenic factors. HuMSC-EV also stimulated vasculogenesis by promoting the recruitment and differentiation of bone marrow progenitors. Moreover, HuMSC-EV improved arteriogenesis by increasing the mobilization of monocytes from the spleen and the bone marrow and their recruitment into the muscle, with a pro-inflammatory potential. Importantly, monocyte depletion by clodronate treatment abolished the proangiogenic effect of HuMSC-EV. The critical role of Ly6C(hi) monocyte subset in HuMSC-EV-induced neovascularization process was further confirmed using Ccr2−/− mice. This study demonstrates that HuMSC-derived EV enhances the neovascularization process in the irradiated tissue by increasing the production of proangiogenic factors, promoting the recruitment of vascular progenitor cells, and the mobilization of innate cells to the injured site. These results support the concept that HuMSC-EV might represent a suitable alternative to stem cells for therapeutic neovascularization in tissue repair.

0.1% ascorbic acid (cat# CC-4176, Lonza). Normal Human Dermal Fibroblasts (NHDF-Ad, CC-2511, Lonza) were cultured with FBM-2 medium supplemented in 2% FBS, 0.1% insulin, 0.1% hFGF-B and 0.1% GA-1000 (CC-3132, Lonza). After thawing, all cells were cultured in 75cm² flasks for amplification, then subcultured in 12-well plates by using Trypsin-EDTA (1X) 0.05% (cat# R001100, Thermo Fisher Scientific, Illkirch, France). Experiments were performed on cultures at passage 5. A scratch injury model was used to investigate the mechanisms of action of EV on the wound healing process. After reaching confluence state, cells were exposed to 20-Gy irradiation and wounds were mechanically made using pipette tip. Cells were washed twice with PBS, and fresh medium supplemented with 50µg of EV, alone or in combination with 10µM of PI3K/AKT inhibitor (LY294002, cat# L9908, Sigma-Aldrich) or TGF-β/SMAD2 inhibitor (SB431542, cat# S4317, Sigma-Aldrich). Cells were incubated and monitored for 48h using a live-cell analysis system (Incucyte® S3,Essen BioScience,Ltd,UK). Pictures of cell cultures were taken every 4h and the area of the wound was analysed using Fiji Software [1].

Real-Time Quantitative Polymerase Chain Reaction
Total RNA and miRNA were extracted from frozen gastrocnemius with mirVana™ miRNA isolation kit (cat# AM1560, Thermo Fisher Scientific) according to the manufacturer's instructions and quantified on a NanoDrop ND-1000 apparatus (NanoDrop Technologies Inc., Rockland, DE). Reverse transcription of miRNA and total RNA was performed using the TaqMan® MicroRNA reverse Transcription kit (cat# 4366596) and the High Capacity Reverse Transcription Kit (cat# 4368814), respectively, according to the manufacturer's instructions (Thermo Fisher Scientific). Quantitative (q)PCR was performed using predeveloped TaqMan® Gene Expression Assays (cat# 4440886, Thermo Fisher Scientific) on an ABI Prism 7900 sequence detection system (Thermo Fisher Scientific). Mouse U6 and GAPDH were used to normalize sample amplification.

Flow cytometry
Mice were euthanized at different times (0, 3, 7, 10, or 14 days) after HuMSC-EV or PBS injection. Peripheral blood was drawn via retro-orbital puncture with heparin solution.
Peripheral blood was lysed after immunofluorescence staining using the FACS lysing solution (cat# 349202, BD Biosciences). BM cells were drawn from femur and filtered through a 40-μm nylon mesh (VWR, Fontenay sous Bois, France). Spleens were collected, gently passed through a 40-μm nylon mesh (VWR). For both splenocytes and BM-derived cells, the cell suspension was centrifuged at 400 x g for 10 min at 4°C. Red blood cells were lysed using red blood cell lysing buffer (cat# R7757, Sigma-Aldrich) and splenocytes and BM cells were washed with PBS.
Muscles were minced with fine scissors, and gently passed through the Bel-Art Scienceware 12-well tissue disaggregator (Thermo Fisher Scientific). Cells were smashed through a 40µm cell strainer (VWR) and harvested in a 50 mL Falcon conical tube. After centrifugation at 400 x g for 15 min at 4°C, cells were resuspended in 100µL of PBS. The total number of cells was then normalized to muscle weight.  5 (e-f) Effect of PI3K/AKT inhibitor LY294002 (e) and TGF-β/SMAD2 inhibitor SB431542 (f) on HuMSC-EV-induced wound healing. Data are means ± SEM (2 independent experiments with triplicate data points, *P<0.05, **P<0.01, ***P<0.001).
Supplementary datafull-length Western blots. Full-length Western blots and annotations are presented as supplementary data at the end of this document. Red arrows indicate the wells and bands that were selected to generate the representative Western blot panels in main figures 1 & 2.